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Short laminin peptide for improved neural stem cell growth.

ABSTRACT: Human neural stem/progenitor cells (hNSCs) are very difficult to culture and require human or animal source extracellular matrix molecules, such as laminin or collagen type IV, to support attachment and to regulate their survival and proliferation. These extracellular matrix molecules are difficult to purify from human or animal tissues, have high batch-to-batch variability, and may cause an immune response if used in clinical applications. Although several laminin- and collagen IV-derived peptides are commercially available, they do not support long-term hNSC attachment and growth. To solve this problem, we developed a novel peptide sequence with only 12 amino acids based on the Ile-Lys-Val-Ala-Val, or IKVAV, sequence: Ac-Cys-Cys-Arg-Arg-Ile-Lys-Val-Ala-Val-Trp-Leu-Cys. This short peptide sequence, similar to tissue-derived full laminin molecules, supported hNSCs to attach and proliferate to confluence for continuous passage and subculture. This short peptide also directed hNSCs to differentiate into neurons. When conjugated to poly(ethylene glycol) hydrogels, this short peptide benefited hNSC attachment and proliferation on the surface of hydrogels and promoted cell migration inside the hydrogels with maximum enhancement at a peptide density of 10 ?M. This novel short peptide shows great promise in artificial niche development for supporting hNSC culture in vitro and in vivo and for promoting hNSC transplantation in future clinical therapy.

SUBMITTER: Li X

PROVIDER: S-EPMC4006481 | biostudies-literature | 2014 May

REPOSITORIES: biostudies-literature

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Short laminin peptide for improved neural stem cell growth.

Li Xiaowei X Liu Xiaoyan X Josey Benjamin B Chou C James CJ Tan Yu Y Zhang Ning N Wen Xuejun X

Stem cells translational medicine 20140401 5

Human neural stem/progenitor cells (hNSCs) are very difficult to culture and require human or animal source extracellular matrix molecules, such as laminin or collagen type IV, to support attachment and to regulate their survival and proliferation. These extracellular matrix molecules are difficult to purify from human or animal tissues, have high batch-to-batch variability, and may cause an immune response if used in clinical applications. Although several laminin- and collagen IV-derived pepti ...[more]

PMID: 24692587

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Project description:Synthetic bioactive hydrogels have been widely recognized as key elements of emerging strategies for tissue engineering. The complex hierarchical structure and chemical composition of the natural ECM inspires us to design multi-component ECM mimics that have potential applications in biomedicine. Taking inspiration from natural proteins, we hypothesized that designing a multi-component synthetic matrix based on short ECM derived peptides will be highly beneficial for providing a functional scaffold, which still remains a challenge in the field of biomaterials. For the proof of concept, we designed a composite hydrogel scaffold inspired by the two essential components of native ECM, i.e. collagen and laminin, which play diverse roles in supporting cell growth. To the best of our knowledge, we have designed the shortest collagen inspired peptide sequence i.e. Nap-FFGSO, which has propensity to self-assemble in the presence of short laminin mimetic peptides. Interestingly, only 10% w/w of laminin peptides was sufficient to provide nucleation and growth of the nanostructures in the collagen inspired peptide and induces further self-assembly to create higher order structures. Such a nucleation and growth mechanism can trigger gelation in the collagen inspired peptides, which otherwise failed to form a gel under physiological conditions. We could achieve similar complexity in the designed matrix through utilization of simple non-covalent interactions, rather than covalent synthetic methodologies to create a dual functional matrix from the non-gelator collagen inspired peptide. The nanofibrous morphology generated through co-assembly can essentially mimic the structure and function of natural ECM, which enables the scaffold to communicate with cells through biochemical signals and promote cell growth, adhesion, proliferation and migration. We envisage that this structural mimicry of a native collagen fibrillar network using such a short peptide sequence can lead to new opportunities for developing next generation functional materials. The strategy of supramolecular assembly using multiple components could develop a plethora of viable biomaterials under physiological conditions.

Project description:Laminin incorporation into biological or synthetic hydrogels has been explored to recapitulate the dynamic nature and biological complexity of neural stem cell (NSC) niches. However, the strategies currently explored for laminin immobilization within three-dimensional (3D) matrices do not address a critical aspect influencing cell-matrix interactions, which is the control over laminin conformation and orientation upon immobilization. This is a key feature for the preservation of the protein bioactivity. In this work, we explored an affinity-based approach to mediate the site-selective immobilization of laminin into a degradable synthetic hydrogel. Specifically, a four-arm maleimide terminated poly(ethylene glycol) (PEG-4MAL) macromer was functionalized with a mono-PEGylated recombinant human N-terminal agrin (NtA) domain, to promote high affinity binding of laminin. Different NtA concentrations (10, 50 and 100 ?M) were used to investigate the impact of NtA density on laminin incorporation, hydrogel biophysical properties, and biological outcome. Laminin was efficiently incorporated for all the conditions tested (laminin incorporation >95%), and the developed hydrogels revealed mechanical properties (average storage modulus (G') ranging from 187 to 256 Pa) within the values preferred for NSC proliferation and neurite branching and extension. Affinity-bound laminin PEG-4MAL hydrogels better preserve laminin bioactivity, compared to unmodified hydrogels and hydrogels containing physically entrapped laminin, this effect being dependent on NtA concentration. This was evidenced by the 10 ?M NtA-functionalized PEG-4MAL gels incorporating laminin that support enhanced human NSC proliferation and neurite extension, compared to the latter. Overall, this work highlights the potential of the proposed engineered matrices to be used as defined 3D platforms for the establishment of artificial NSC niches and as extracellular matrix-mimetic microenvironments to support human NSC transplantation.

Project description:UnlabelledHuman neural stem/progenitor cells (hNSPCs) are good candidates for treating central nervous system (CNS) trauma since they secrete beneficial trophic factors and differentiate into mature CNS cells; however, many cells die after transplantation. This cell death can be ameliorated by inclusion of a biomaterial scaffold, making identification of optimal scaffolds for hNSPCs a critical research focus. We investigated the properties of fibrin-based scaffolds and their effects on hNSPCs and found that fibrin generated from salmon fibrinogen and thrombin stimulates greater hNSPC proliferation than mammalian fibrin. Fibrin scaffolds degrade over the course of a few days in vivo, so we sought to develop a novel scaffold that would retain the beneficial properties of fibrin but degrade more slowly to provide longer support for hNSPCs. We found combination scaffolds of salmon fibrin with interpenetrating networks (IPNs) of hyaluronic acid (HA) with and without laminin polymerize more effectively than fibrin alone and generate compliant hydrogels matching the physical properties of brain tissue. Furthermore, combination scaffolds support hNSPC proliferation and differentiation while significantly attenuating the cell-mediated degradation seen with fibrin alone. HNSPCs express two fibrinogen-binding integrins, αVβ1 and α5β1, and several laminin binding integrins (α7β1, α6β1, α3β1) that can mediate interaction with the scaffold. Lastly, to test the ability of scaffolds to support vascularization, we analyzed human cord blood-derived endothelial cells alone and in co-culture with hNSPCs and found enhanced vessel formation and complexity in co-cultures within combination scaffolds. Overall, combination scaffolds of fibrin, HA, and laminin are excellent biomaterials for hNSPCs.Statement of significanceInterest has increased recently in the development of biomaterials as neural stem cell transplantation scaffolds to treat central nervous system (CNS) injury since scaffolds improve survival and integration of transplanted cells. We report here on a novel combination scaffold composed of fibrin, hyaluronic acid, and laminin to support human neural stem/progenitor cell (hNSPC) function. This combined biomaterial scaffold has appropriate physical properties for hNSPCs and the CNS, supports hNSPC proliferation and differentiation, and attenuates rapid cell-mediated scaffold degradation. The hNSPCs and scaffold components synergistically encourage new vessel formation from human endothelial cells. This work marks the first report of a combination scaffold supporting human neural and vascular cells to encourage vasculogenesis, and sets a benchmark for biomaterials to treat CNS injury.

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Short laminin peptide for improved neural stem cell growth.

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Short laminin peptide for improved neural stem cell growth.

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